Antibiotic peptides from bovine milk

ABSTRACT

A peptide having the amino acid sequence 
     H 2 N—X 1 —R—X 3 —X 2 —COOH  (formula I) 
     wherein  
     X 1  is either zero or  
     X 1  and/or X 2  are a residue representing at least five amino acid residues (symbolized in the one letter amino acid code), preferably naturally occurring amino acids,  
     with the proviso that  
     X 1  and/or X 2  contain at least one basic amino acid residue immediately followed by a hydrophobic amino acid residue and X 1  and/or X 2  contain at least one glutamine residue.

[0001] The present invention is concerned with peptides having the aminoacid according to claim 1, fragments of the peptides obtainable byproteolytic cleavage, a medicament comprising a peptide of theinvention, a process for the manufacturing of peptides of the invention,a method of treating by ad-ministering a peptide of the invention aswell as methods of using the peptides of the invention.

[0002] Antimicrobial peptides are small peptide compounds which arecapable to decrease the incidence of disease and could serve asmodulators of a naturally occurring bacterial flora (Ganz T. et al.,1992, Med. Microbiol. Immunol. 181, 99-105; Eisenhauer P.B. et al.,1992, Infect. Immun. 60, 3556-3565; Oulette A. J. et al., 1992, FEBSLett. 304, 146-148; Jones D. E. and Bevins C. L., 1993, FEBS Lett. 315,187-192; Selstedt M.E. et al. (1992) J. Cell. Biol. 118, 929-936).

[0003] The fact that milk could influence micro-organisms is also wellestablished (Wharton B. A. et al, 1994, Acta Paediatr. Jpn. 36,579-584). Present factors thought to be responsible are lactoferrin(Bullen J.J. et al., 1972, Brit. J. Med. 1, 69-72; Baggiolini M. et al,1970, J. Exp. Med. 13, 559-570), lysozyme (Fleming A., 1922, Proc. Roy.Soc., London, 93, 306-317, Jolles J. and Jolles P., 1968, Bull. Soc.Chim. Biol., Paris, 50, 2543-2551), lactoperoxidase (Cals M. M. et al.,1991, Eur. J. Biochem. 198, 733-739, Bullen J. J. et al., 1972, Brit. J.Med. 1, 69-72). Milk is a rich source of peptides mainly derived fromproteolytic cleavage of proteins. Beyond a nutritive value severalbiological effects were described like immunomodulation, antithromboticactivities, opioid action or inhibition or mineral carriage (Meisel H.et al., 1989, Z. Ernahrungswiss. 28, 267-278; Fiat A. M. and Jolles P.,1989, Mol. Cell. Biochem. 87, 5-30; Fiat A. M. et al., 1993, J. Dairy.Sci. 76, 301-310).

[0004] Casocidin-I is a peptide compound, a defined cleavage productfrom casein-alpha-s2, which is naturally occurring in bovine milk as itcould be purified in authentic form (Zucht H.D., 1995, FEBS Lett. 372,185-188). It has no counterpart in human milk since human milk does notcontain any casein of the alpha-s2 type.

[0005] Surprisingly, the mild antibacterial effect found in milk couldbe enriched several orders of magnitude by the peptides of theinvention. According to the invention a peptide with antibiotic effectsshow the amino acid sequence

H₂N—X₁—R—X₂—COOH  (formula I)

[0006] wherein

[0007] X₁ is either zero or

[0008] X₁ and/or X₂ are a residue representing at least five amino acidresidues (symbolized in the one letter amino acid code) preferablynaturally occurring amino acids,

[0009] with the proviso that

[0010] X₁ and/or X₂ contain at least one basic amino acid residueimmediately followed by a hydrophobic amino acid residue and X₁ and/orX₂ contain at least one glutamine residue.

[0011] Preferably, the distance of the hydrophobic residue and R shouldnot exceed 7-10 amino acids.

[0012] It has now been found that these peptides could kill a broadrange of microorganisms with a preference of microorganisms notnaturally present in the normal intestinal flora. Surprisingly, thepeptides of the invention show activity also against eukaryoticorganisms such as fungi but do not effect other eukaryotic cells as forexample HT 29 cells. The peptides of the invention are therapeuticaltools for treating diseases caused by pathogenic eukaryots, for example,protists which are only present in the adult cattle intestine and thatof man respectively.

[0013] In a preferred embodiment the peptide of the invention has astructure wherein

[0014] X₁ represents —X_(m)—K—B—X_(n)— wherein

[0015] X is any amino acid residue having at least one Q,

[0016] B is any hydrophobic amino acid residue;

[0017] m=0−40, n=0−8 and

[0018] X₂ is Q.

[0019] A further preferred embodiment of the peptide of the invention isa structure wherein

[0020] X₁ is zero or represents Q,

[0021] X₂ represents —X_(n)—K—B—X_(m)—,

[0022] X, B, m and n having the same meaning as defined above.

[0023] A further preferred embodiment of the present invention is apeptide having a structure wherein

[0024] X₁ represents —X_(m)—K—B—X_(n)— and

[0025] X₂ represents —X_(n)—K—B—X_(m),

[0026] X,B,m, and n have the same meanings as defined above.

[0027] It is understood by the skilled person that not only aminonaturally occurring can be used in effective peptides. Therefore, theamino acids in the chain forming the peptides can be modified in orderto match valuable properties for respective use. For example, it ispossible to use mixtures of D- and/or L-amino acids in the amino acidsequence of the peptides of the invention in order to modify thepharmaceutical effects.

[0028] Furthermore, there may be introduced modification in the sidechains of the peptide backbone of the amino acid sequence. For example,it is possible to introduce sugar moieties at suitable positions, forexample, asparagine. Glycosylation modifies affinity of the peptides orits solubility. It may also be useful to phophorylate amino acids of thepeptide of the invention. It may also be useful to modified amino acidside chains in order to modify the hydrophobicity or hydrophilicity ofthe peptide. For example, if the peptide is used in a pharmaceuticalcomposition which is designed for topical application it may beadvantageous to modify the peptide that it more easily penetrates theskin in order to develop its activity also in deeper layers of the skin.On the other hand if only a surface treatment is needed the propertiesof the peptide could be modified in so far as it is not penetrating toofar into the deeper layers of the skin.

[0029] A further modification may occur at the N-terminal or C-terminalend of the peptide. The N-terminal end may be modified withelectrophilic agents. For example, the N-terminal amino group may bealkylated, acylated, cleaved off or protected. The N-terminal end may beprotected with labile chemical groups which are cleaved off during theuse of the peptide. The C-terminal end of the peptides can be modifiedwith nucleophilic reagents in order to attack the carbonyl group of thecarboxyl and the C-terminal end may be modified, for example byamidation, esterification and the like.

[0030] It is possible to obtain peptides by proteolytic cleavage of thepeptides of the invention. Such fragments which fall into the scope ofthe invention must show antifungal activity after protease treatment.Suitable proteases for obtaining fragments from the peptides of theinvention are endo-proteinases like trypsin, subtilisin, thermolysin andendo-proteinase LYS-C.

[0031] Preferably, the peptide of the invention consists of a domain-RYQ-. However, for example, a tetrapeptide with the motif QRYQ alone isno t s sufficient to gain anti microbial properties.

[0032] A further preferred structure on both sides of this core motif-RYQ- is the occurrence of one or more basic residues immediatelyfollowed by a hydrophobic amino acid residue. The specificity of thepeptides of the invention can be modulated by composition of theresidues flanking the core region -RYQ- so that the detailedantimicrobial spectrum of the peptide of the invention could be finetuned by means of modulating residues around the core motif.

[0033] Within the side chain one or more glutamine residues seem to beof some relevance since the preferred peptide casocidin-I (casocidin1-39) contains high an amount of glutamine residues. On the other sidethe glutamine residues alone do not promote the antimicrobial effect ascasein-alpha-s2-80-120 shows a very similar glutamine pattern but doesnot exhibit any antibacterial effects.

[0034] Specifically preferred peptides according to the inventions arethose as follows:

[0035] Casocidin-I

[0036] KTKLTEEEKNRLNFLKKISQRYQKFALPQYLKTVYQHQK (Casocidin 1-39)

[0037] TKLTEEEKNRLNFLKKISQRYQKFALPQYLKTVYQHQK (Casocidin 2-39)

[0038] KNRLNFLKKISQRYQKFALPQYLKTVYQHQK (Casocidin 9-39)

[0039] RYQKFALPQYLKTVYQHQK (Casocidin 21-39)

[0040] KNRLNFLKKISQRYQ (Casocidin 9-23).

[0041] As the skilled person understand also this very specific peptidescan be modified according to the strategies outlined above discussingthe general aspects of the invention.

[0042] According to the invention the process for the manufacturing of apeptide according to the invention comprises the steps of

[0043] treating bovine milk with acetic acid and calcium sulfate,

[0044] heating the mixture obtained

[0045] removing the precipitate and isolating the supernatant,

[0046] treating the supernatant with a cation-exchanger resin forbinding of basic peptides, washing the resin optionally with a ureasolution to remove non specifically bound material,

[0047] isolating the resin having peptides bound to it,

[0048] elution of peptides bound to the resin and further purificationwith chromatographic operations,

[0049] optionally followed by fragmentation, e.g. by digestion withproteases.

[0050] The precise conditions are exemplified in the working examples ofthe specification herein below. The biological activity of the peptidesof the invention show a broad range specificity against grampositive andgramnegative bacteria. They are also capable to inhibit the growth offungi exemplified on Rhodotorula rubra. Intestinal cells were notkilled. This shows that they are specificly effective against thepathogene but not against the host. Preferably, the activity ispronounced in low ion strength media in the absence of high amounts ofcalcium. It is preferred to administer the peptide of the invention in ahypotonic medium in the presence of calcium complexing agents such ascitrate, phosphate, EGTA in order to facilitate its activity. Themechanism of action may be due to the membrane permeabilizing effect ofthe peptide depending on the dosage of the peptides of the invention.

[0051] The peptides of the invention can be used for formulating amedicament for treating diseases which are caused by bacteria or fungi.The medicament optionally contains further active compounds or carriermaterials and or adjuvants. In a preferred embodiment of the presentinvention the medicament of the invention is formulated for topicaladministration or administration on mucosa such as the mucosa of mouthand vagina. Because of its antifungal potency the medicament of thepresent invention is preferably useable for treating fungal diseases andor treatment of diseases which are caused by eukaryotic organisms suchas tricho-monades, for example in vagina, or organisms such as filariaor plasmodium.

[0052] The peptide of the invention preferably casocidin-I can beadministered in the form of an oral dosage unit together with fillerswhich are pharmaceutically acceptable. It can be used in liquidpreparations or in a dry preparation with lyo-philized peptide. Thetypical dosage would be in the range of 1 μg/ml up to 250 μg/ml of themedicament. Casocidin-I keeps its potency when lyophilized and can bestored at room temperature in this form. It allows long term storagewith reconstitution with an appropriate low ion strength buffer, forexample, 25 mOsm prior to use. A supplementation with pharmaceuticallyacceptable agents capable to complex calcium like citrate (about 5-250mM), phosphate (5-150 mM) or EGTA (1 mM) support the antimicrobialeffect of the drug.

[0053] If antimicrobial diseases of the skin have to be treated theformulation preferably is in the form of an ointment. The concentrationrange of the drug in such a formulation is comparable to the abovementioned range of 0,1 ng/ml to 1 mg/ml, preferably 1 μg/ml to 250μg/ml. The dosis can be adjusted obeying the rules of the art as canreadily understood by the skilled person.

[0054] The peptides of the invention can be used for therapy of diseasessuch as diarrhoea, diseases caused by a change of the microbiologicalproperties or conditions in the intestine, the mucosa of the vagina orthe mouth, diseases of the skin and diseases caused by fungi.

[0055] The peptide of the invention can be used as preservative of foodor perishable goods or as adjuvant for fermentation processes.

[0056]FIG. 1

[0057] Purification of casocidin-I: (a) RP-C18 HPLC of the peptideseluted from the cation exchanger. The growth inhibitory activity wasmonitored with E. coli in the radial diffusion assay. (b) RP-C18 HPLCrechromatography of the most active fraction. (c) RP-C4 HPLCrechromatography of fraction 13. (d) CZE analysis of the purificationproduct casocidin-I.

[0058]FIG. 2

[0059] Effect of casocidin-I on the CFU of E. coli BL21 depending onincubation time and dosage. The picture shows various plates withbacterial colonies.

[0060]FIG. 3

[0061] Dose response curves of casocidin-I with diverse microorganismsin the preincubation experiment (determination of CFU).

[0062]FIG. 4

[0063] Radial diffusion experiment, indicating the antimicrobialactivity of casocidin-I.

[0064]FIG. 5

[0065] Release of intracellular enzymes beta-lactamase andbeta-galactosidase indicating the effect of casocidin-I.

[0066] The purification of casocidin-I out of bovine milk is exemplifiedas follows.

[0067] Grade A bovine milk is to be used. In order to denature most ofthe high molecular weight proteins, the milk must be initially treatedby boiling for 5 min and acidifying with 10% (v/v) acetic acid.Additionally, 1 g/l calcium chloride (CaCl₂*2 H₂O) is added toprecipitate calcium-dependent phosphoproteins. After centrifugation (15min, 3,500×g, and 4° C.) the resulting supernatant is collected, dilutedwith 4 volumes of water and incubated with a strong cation exchangeresin (Parcomer, 20 μm, Biotek, Heidelberg, Germany).

[0068] The resin is washed with 5 M urea in 5 mM phosphate buffer (pH3.0) and with an excess of water. To elute bound peptides the resin istreated with 1 M NaCl in 5 mM phosphate buffer (pH 3.0) at roomtemperature. The eluate is centrifuged (5 min, 1,000×g, RT) to removeresidual resin particles.

[0069] The HPLC purification steps were performed on Parcosil-C₁₈ (1cm×12.5 cm, 100 Å, 5 μm) and Parcosil-C₄ (0.4 cm×12.5 cm, 300 Å, 5 μm)reversed phase columns (Biotek, Heidelberg, Germany). Afterequilibration with 0.1% trifluoroacetate (TFA), peptides are eluted bylinearly increasing the amount of solvent B (acetonitrile with 0.1%TFA). For the first separation step a Parcosil C₁₈ RP column (1 cm×12.5cm, 100 Å, 5 μm) is used with the following gradient: 0-50 min, 0-80% B.The bactericidal activity of an aliquot were monitored with the radialdiffusion assay and the maximum activity was pooled. The second step ofpurification was performed with the same RP-C₁₈ column using a lesssteep gradient (gradient 2:0-5 min, 0-15% B; 5-65 min, 15-80% B) (FIG.2). In the third step of purification, the antibiotic activity offraction 13 could be recovered by RP-C₄ HPLC using the followinggradient (0-5 min, 0-15% B; 5-65 min, 15-80% B) . Fraction 8 containscasocidin-I with a relative high purity. The purification is illustratedin FIG. 1 a-d.

[0070] The peptide analysis revealed the following structure ofcasocidin-I:

[0071] KTKLTEEEKNRLNFLKKISQRYQKFALPQYLKTVYQHQK

[0072] SYNTHESIS OF CASOCIDIN-I

[0073] Chemical synthesis of peptides was carried out as describedelsewhere (Atherton E. and Sheppard R.C., 1989, Solid phase peptidesynthesis, IRL Press, Oxford; Jones, J., 1991, The chemical synthesis ofpeptides, Clarendon Press, Oxford). The purification was performed on aVydac RP-C₁₈ column (MZ-Analysentechnik, Mainz, Germany, 10 μm, 300 Å, 2cm×25 cm) with 0.06% TFA/acetonitrile/water at a flow rate of 10 ml/min.The purity was controlled by means of HPLC and mass spectrometry.

[0074] ANTIMICROBIAL ASSAYS

[0075] BACTERIA

[0076] E. coli Xl1 blue is a commercial strain distributed by Stratagene(USA). E. coli BL 21 (DE3) was a gift from A. Ebneth (Medical SchoolHannover, Dept. of Biophysics). Staphylococcus carnosus T300 was a giftfrom F. Goetz (Freiburg). Rhodotorula rubra, Staph. epidermidis is aclinical isolate from I. Zimmer and M. Weimann (laboratory of ClinicalMedicine, Hannover). All other bacteria were purchased at DeutscheSammlung von Mikroorganismen und Zellkulturen GmbH (DSM, Braunschweig,Germany). All bacteria were routinely cultured on LB-Broth (10 g/lcaseinhydroly-sate, 5 g/l yeast extract, 5 g/l NaCl, pH 7.4).

[0077] PREINCUBATION EXPERIMENT

[0078] Exponentially grown bacteria with a density corresponding to anA₆₀₀ of 0.4 to 0.6, were initially diluted 1: 10⁵ in 10 mM phosphatebuffer (Na—P, pH 7.2) or phosphate buffered saline (PBS, pH 7.4) andincubated at 37° C. for 120 min in Na—P or PBS supplemented withdifferent concentrations of peptides. After this period the reactionmixtures were diluted 1:10 and 1:100 in the same buffer and plated onLB-Agar to determine the colony forming units (CFU). The plating weredone in triplets. The colonies were counted with a colony counter(Darkfield, Quebec, Canada).

[0079] An example for the depletion of viable bacteria gives FIG. 2showing that a time dependent killing of the bacteria during incubationoccurs. Further results of preincubation experiments are summarized inFIG. 3. The interpolation of the date gives the following order ofsensitivity of the used strains B. subtilis>S. carnosus=S.epidermidis>Rhodoto-rula >>E. coli>Enterococcus faecium ATCC 35667. Theconcentration necessary to reduce the colony forming units (CFU) to 50%of the initial CFU ranges from 180 μg/ml (E. faecium) over 20 μg/ml (E.coli, S. carnosus, R. rubra) to 1 μg/ml for B. subtilis indicating apotent inhibitory effect of the used peptide.

[0080] RADIAL DIFFUSION EXPERIMENT

[0081] The radial diffusion assay performed as described in (Lehrer R.I.et al., 1991, J. Immun. Meth. 137, 167-173) with slight modifications.Bacteria were grown to a final absorbance at 580 nm of 0.2 (appr.2×10¹⁰/ml). The agarose layer contained 30 mg/100 ml tryptic soy broth(TSB, Sigma chemicals, Germany) in 10 mM sodiumphosphate 10 mM pH 7.2with 0.02% Tween® 20 and 0.6% agarose without EEO (Serva, Heidelberg,Germany) . One ml of the bacteria suspension was added to 50 ml agaroseat 50° C. and the plates were poured immediately. Afterwards the plateswere incubated at 4° C. to harden the agarose. The bacteria wereincubated for 10 hours at 37° C. with the peptide substrate applied intoholes of 3 mm diameter in the agarose layer. In some cases the plateswere first fixed with 0.1% glutardialdehyde and afterwards stained withGiemsa stain (Merck, Darmstadt, Germany) diluted in 10 mM Tris-HCl pH7.2 and washed with an excess of the same Tris Buffer. Using Rhodotorularubra the medium contained 0.8% low melting point agarose (Sigma,Germany), 0.5 g/ 100 ml TSB and 20 mM TRIS-HCl pH 7.5. The yeast wasadded after an equilibration of the medium at 37° C. and poured into theplates. The incubation was performed for 10 h at 30° C.. The inhibitoryinfluence of casocidin-I shows FIG. 4.

[0082] INFLUENCE OF MEDIUM STRENGTH AND CALCIUM IONS

[0083] To determine the minimal inhibitory concentration of casocidin-Iunder the influence of the medium strength or in the presence ofbivalent cations, a dilution assay was performed in 96-well cell cultureclusters. After diluting the peptide in 50 μl medium (tryptic soy broth,Sigma, Germany) with adjusted pH 7.2, ion concentrations or mediumstrength on one plate, the same amount of medium with appr. 2-4 * 10³bacteria in 50 μl medium were added to the diluted samples. Theincubations were done overnight at 37° C.. The minimal inhibitoryconcentration was manually read using a microscope. TABLE Example of theeffect of the medium strength and bivalent cations on S. carnosus Mediumstrength 1 0.5 0.2 0.1 0.05 0.02 MIC μg/ml no 25 3.3 3.3 1.7 0.6bivalent Cation 2.5 mM Mg  10 mM Mg 2.5 mM Ca 2.5 mM Mn MIC μg/ml  20100 no no

[0084] HT 29 (human colon carcinoma) cells were grown as monolayers at37° C. in RPMI 1640 medium (Gibco, Germany) containing 2 mM L-glutamine,100 U/ml penicillin, 0.1 mg/ml streptomycin and 10% heat inactivatedfetal calf serum in 5% CO₂ on 24-well tissue culture clusters (Nunc,Germany) by loading of a suspension of 5 * 10⁵ cells/ml. To determinethe cytotoxic effect of casocidin-I, HT 29 cells propagated in tissueculture clusters were incubated with different concentrations ofcasocidin-I (from 1 μg/ml to 100 μg/ml) for up to 24 h at 37°. Theexperiments show no cytotoxic effects to this intestinal cell line.

[0085] DETERMINATION OF THE MEMBRANE PERMEABILIZATION

[0086] To determine the mechanism of action of casocidin-I which is amembrane permeabilizing effect we determined the release ofintracellular enzymes beta-galactosidase and beta-lacta-mase from E.coli transformed with the plasmid pUC18 (FIG. 5). The beta-galactosidaseindicated the degree of the permeabilization of the inner membrane (IM)of E. coli. The permeabilization of the outer membrane was monitored bydetermination of released beta-Lactamase (plasmid coded) mainly presentin the periplasmatic space.

[0087] To measure the relative release of beta-galactosidase asuspension of E. coli grown overnight in LB medium was washed once inphosphate buffer (10 mM, pH 7.0). Afterwards the bacteria wereresuspended to a final density of app. 0.5 A₆₀₀. Peptide fractions werelyophilized in 1.5 ml tubes in a speed vac concentrator and solved in 40μl phosphate buffer. To the peptide solution a volume of 200 4μlbacterial suspension was added and the incubation was performed at 37°C. for 30 min. As a control, to determine the maximum of the enzymerelease, bacteria were disrupted by ultrasonic treatment (about 100joules in 10 sec). The samples were afterwards centrifuged for 5 min at15,000×g at 4° C. and the supernatant immediately removed from thesediment. To detect the beta-galactosidase activity an amount of 40 μllysate was mixed with 460 μl of the detection reagent (60 mM Na₂HPO₄,400 mM NaH₂PO₄, 10 mM KCl, 1 mM MgSO₄, 50 mM beta-mercaptoethanol, 0.89mg/ml o-nitro phenyl galactoside ONPG) and incubated at 37° C. for 45min. The absorbance of the samples at 405 nm was measured and theincrease of each absorbance was normalized to the sonified sampleassuming enzymatic release of 100%.

[0088] To detect the release of beta-lactamase the supernatants of E.coli were prepared as described for the beta-galactosi-dase. As washingbuffer PBS was used. To monitor the beta-lactamase activity an amount of50 μl. supernatant was mixed with 50 μM PADAC (Calbiochem, USA) in 10 mMNa—P pH 7.2. The samples were incubated for 45 min at 37° C. in the darkand the absorbance was measured at a wavelength of 700 nm and thenormalization was done with the results gained with sonified samples.

[0089] PEPTIDE ANALYSIS

[0090] Amino acid sequence determinations were carried out automaticallyon an Applied Biosystems 473 A gas phase sequencer (Applied BiosystemsDiv. of Perkin Elmer, Weiter-stadt, Germany). Capillary zoneelectrophoresis (CZE) was performed with a 50 cm uncoated capillary offused silica on the CZE system model P/ACE 2100 (Beckmann, Munchen,Germany). The running buffer used was 100 mM NaH₂PO₄, pH 2.5 containing0.02% hydroxypropyl methyl cellulose. After injection of 60 nl sample,the separation was carried out with a constant current of 120 μA. Massspectrometric analysis (MS) was performed on a triple-stage quadrupoleelectrospray mass spectrometer Sciex API III (Perkin Elmer, Ueberlingen,Germany) equipped with an articulated ion-spray source operation atatmospheric pressure. The samples were diluted in 50% acetonitrile, 0.2%acetic acid prior to injection. Mass spectra were recorded in positiveion mode.

1 5 1 39 PRT Bovine sp. 1 Lys Thr Lys Leu Thr Glu Glu Glu Lys Asn ArgLeu Asn Phe Leu Lys 1 5 10 15 Lys Ile Ser Gln Arg Tyr Gln Lys Phe AlaLeu Pro Gln Tyr Leu Lys 20 25 30 Thr Val Tyr Gln His Gln Lys 35 2 38 PRTBovine sp. 2 Thr Lys Leu Thr Glu Glu Glu Lys Asn Arg Leu Asn Phe Leu LysLys 1 5 10 15 Ile Ser Gln Arg Tyr Gln Lys Phe Ala Leu Pro Gln Tyr LeuLys Thr 20 25 30 Val Tyr Gln His Gln Lys 35 3 31 PRT Bovine sp. 3 LysAsn Arg Leu Asn Phe Leu Lys Lys Ile Ser Gln Arg Tyr Gln Lys 1 5 10 15Phe Ala Leu Pro Gln Tyr Leu Lys Thr Val Tyr Gln His Gln Lys 20 25 30 419 PRT Bovine sp. 4 Arg Tyr Gln Lys Phe Ala Leu Pro Gln Tyr Leu Lys ThrVal Tyr Gln 1 5 10 15 His Gln Lys 5 15 PRT Bovine sp. 5 Lys Asn Arg LeuAsn Phe Leu Lys Lys Ile Ser Gln Arg Tyr Gln 1 5 10 15

1. A peptide having the amino acid sequence H₂N—X₁—R—X₂—COOH  (formulaI) wherein X₁ is either zero or X₁ and/or X₂ are a residue representingat least five amino acid residues (symbolized in the one letter aminoacid code), preferably naturally occurring amino acids, with the provisothat X₁ and/or X₂ contain at least one basic amino acid residueimmediately followed by a hydrophobic amino acid residue and X₁ and/orX₂ contain at least one glutamine residue.
 2. The peptide according toclaim 1 wherein X₁ represents —X_(m)—K—B—X_(n)— wherein X is any aminoacid residue having at least one Q. B is any hydrophobic amino acidresidue; m=0-40, n=0-8 and X₂ is Q.
 3. The peptide according to claim 1wherein X₁ is zero or represents Q. X₂ represents —X_(n)—K—B—X_(m)—, X,B, m and n having the same meaning as defined in claim
 2. 4. The peptideaccording to claim 1 wherein X₁ represents —X_(m)—K—B—X_(n)— and X₂represents —X_(n)—K—B—X_(m), X,B,m, and n have the same meanings asdefined in claim
 2. 5. The peptide according to any one of claims 1-4,having D- and/or L-amino acids.
 6. The peptide according to anyone ofthe claims 1-5, having the structuresKTKLTEEEKNRLNFLKKISQRYQKFALPQYLKTVYQHQK (Casocidin 1-39)TKLTEEEKNRLNFLKKISQRYQKFALPQYLKTVYQHQK (Casocidin 2-39)KNRLNFLKKISQRYQKFALPQYLKTVYQHQK (Casocidin 9-39) RYQKFALPQYLKTVYQHQK(Casocidin 21-39) KNRLNFLKKISQRYQ (Casocidin 9-23).
 7. The peptideaccording to anyone of the claims 1-6 being chemically modified in theamino acid side chains and/or the amino group of the N-terminal end hasbeen modified with electrophilic reagents and/or the carboxyl moiety ofthe C-terminal end of the peptide has been modified with nucleophilicreagents.
 8. Fragments of the peptides according to anyone of the claims1-7, obtainable by proteolytic cleavage with proteases, which fragmentsshow antifungal and/or antibiotic effects.
 9. A medicament comprising atleast one of the peptides according to any one of the claims 1-8 in aneffective amount for treating diseases, optionally in a mixture withfurther active compounds and or carrier materials or adjuvants.
 10. Themedicament of claim 9 formulated for topical administration oradministration on mucosa.
 11. The medicament of claims 9 for treating offungal diseases, and/or treatment of diseases caused by eukaryoticorganisms such as trichomonades in vagina, or organisms as filaria orplasmodium.
 12. A process for the manufacturing of a peptide accordingto claim 1-8 comprising the steps treating bovine milk with acetic acidand calcium sulfate, heating the mixture obtained removing theprecipitate and isolating the super- natant, treating the supernatantwith a cation-exchanger resin for binding of basic peptides, washing theresin optionally with a urea solution to remove non specifically boundmaterial, isolating the resin having peptides bound to it, elution ofpeptides bound to the resin and further purification withchromatographic operations, optionally followed by fragmentation, e.g.by digestion with proteases.
 13. A method of treating of diseases byadministering a medicament of claim 9 or 11 in an effective amount to apatient in need thereof.
 14. The method of claim 13 wherein the diseaseis diarrhoea, diseases caused by a change of the microbiologicalproperties or conditions in the intestine, the mucosa of the vagina orthe mouth, diseases of the skin and diseases caused by fungi.
 15. Methodof using a peptide according to any one of the claims 1-8. aspreservative of food or perishable goods or as adjuvant for fermentationprocesses.
 16. Method of using a peptide according to any one of theclaims 1-8 for the manufacturing of a medicament according to claims9-11.
 17. A peptide having the amino acid sequenceH₂N—X₁—R—X₂—COOH  (formula I) wherein, X₁ is either zero or X₁ and/or X₂are a residue representing at least five amino acid residues (symbolizedin-the one letter amino acid code), preferably naturally occurring aminoacids, with the proviso that X₁ and/or X₂ contain at least one basicamino acid residue immediately followed by a hydrophobic amino acidresidue and X₁ and/or X₂ contain at least one glutamine residue. withthe proviso that X₁ does not represent: KTKLTEEEKNRLNFLKKISQ X₂ does notrepresent: TKLTEEEKNRLNFLKKISQ.
 18. The peptide according to claim 17wherein X₁ represents —X_(m)—K—B—X_(n)— wherein X is any amino acidresidue having at least one Q. B is any hydrophobic amino acid residue;m=0-40, n=0-8 and X₂ is Q.
 19. The peptide according to claim 17 whereinX₁ is zero or represents Q. X₂ represents —X_(n)—K—B—X_(m)—, X, B. m andn having the same meaning as defined in claim
 18. 20. The peptideaccording to claim 17 wherein X₁ represents —X_(m)—K—B—X_(n)— and X₂represents —X_(n)—K—B—X_(m), X,B,m, and n have the same meanings asdefined in claim
 18. 21. The peptide according to any one of claims17-20, having D- and/or L-amino acids.
 22. The peptide according toanyone of the claims 17-21, having the structuresKNRLNFLKKISQRYQKFALPQYLKTVYQHQK (Casocidin 9-39) RYQKFALPQYLKTVYQHQK(Casocidin 21-39) KNRLNFLKKISQRYQ (Casocidin 9-23).
 23. The peptideaccording to anyone of the claims 17-22 being chemically modified in theamino acid side chains and/or the amino group of the N-terminal end hasbeen modified with electrophilic reagents and/or the carboxyl moiety ofthe C-terminal end of the peptide has been modified with nucleophilicreagents.
 24. Fragments of the peptides according to anyone of theclaims 17-23, obtainable by proteolytic cleavage with proteases, whichfragments show antifungal and/or antibiotic effects.
 25. A medicamentcomprising at least one of the peptides according to any one of theclaims 17-24 in an effective amount for treating diseases, optionally ina mixture with further active compounds and or carrier materials oradjuvants.
 26. The medicament of claim 25 formulated for topicaladministration or administration on mucosa.
 27. The medicament of claims25 for treating of fungal diseases, and/or treatment of diseases causedby eukaryotic organisms such as trichomonades in vagina, or organisms asfilaria or plasmodium.
 28. A process for the manufacturing of a peptideaccording to claim 17-24 comprising the steps treating bovine milk withacetic acid and calcium sulfate, heating the mixture obtained removingthe precipitate and isolating the super-natant, treating the supernatantwith a cation-exchanger resin for binding of basic peptides, washing theresin optionally with a urea solution to remove non specifically boundmaterial, isolating the resin having peptides bound to it, elution ofpeptides bound to the resin and further purification withchromatographic operations, followed by fragmentation, e.g. by digestionwith proteases.
 29. A method of treating of diseases by administering amedicament of claim 25 or 27 in an effective amount to a patient in needthereof.
 30. The method of claim 29 wherein the disease is diarrhoea,diseases caused by a change of the microbiological properties orconditions in the intestine, the mucosa of the vagina or the mouth,diseases of the skin and diseases caused by fungi.
 31. Method of using apeptide according to any one of the claims 17-24 as preservative of foodor perishable goods or as adjuvant for fermentation processes. 32.Method of using a peptide according to any one of the claims 17-24 forthe manufacturing of a medicament according to claims 25-27.